DESCRIPTION The long-term goal of the proposed research is to understand the physiological and developmental roles of nebulin isoforms in vertebrate skeletal muscles, which have been identified by variability in the molecular mass (600-900 kD). This giant modular protein has been proposed to act as a protein ruler to regulate the length of thin filaments, as a calcium-calmodulin mediated regulatory protein of actomyosin interaction, and as a template for the assembly of thin filament lattice during development. The specific aims are: (1) to characterize nebulin isoforms in a wide range of skeletal muscles of fetal, neonatal and adult rabbits and rats. Nebulin expression will be compared and contrasted with the transition of other myofilament proteins such as titin, myosin, C-protein, actin, tropomyosin, troponin, alpha-actin, Cap Z and tropomodulin; (2) to identify and prepare isoform-specific and pan anti-nebulin antibodies. Epitope profiles of monoclonal anti-nebulin will be characterized for each isoform. Anti-peptide antibodies will be produced to common and variable regions of nebulin sequences; (3) to correlate the size of nebulin isoforms and the length of actin filaments in selected fetal, neonatal and adult skeletal muscles of rabbit and rats. Scanning transmission electron microscopy and mass spectroscopy will be used to determine the size and contour length of nebulin isoforms. Immunolocalization of nebulin isoforms, actin, tropomyosin, tropomodulin and cap Z will be used to determine the cellular distribution and length and span of thin filaments; and (4) to characterize the molecular basis of nebulin isoform diversity. Developmentally regulated expression of nebulin exons will be determined by polymerase chain reaction and nucleotide sequencing. The proposed research would define the physiological function of nebulin and the developmental and physiological basis for its contribution to the structure, regulation, and assembly of thin filaments in skeletal muscles in healthy and diseased states.